Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum

J Physiol. 2005 May 1;564(Pt 3):737-49. doi: 10.1113/jphysiol.2005.085035. Epub 2005 Feb 24.

Abstract

Unloading of endoplasmic reticulum (ER) Ca(2+) stores activates influx of extracellular Ca(2+) through 'store-operated' Ca(2+) channels (SOCs) in the plasma membrane (PM) of most cells, including astrocytes. A key unresolved issue concerning SOC function is their spatial relationship to ER Ca(2+) stores. Here, using high resolution imaging with the membrane-associated Ca(2+) indicator, FFP-18, it is shown that store-operated Ca(2+) entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane-ER junctions. In the absence of extracellular Ca(2+), depletion of ER Ca(2+) stores using cyclopiazonic acid, an ER Ca(2+)-ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca(2+) concentration ([Ca(2+)](SPM)) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca(2+) causes localized Ca(2+) influx that first increases [Ca(2+)](SPM) in the same restricted regions and then, with a delay, in ER-free regions. Antisense knockdown of the TRPC1 gene, proposed to encode endogenous SOCs, markedly reduces SOCE measured with Fura-2. High resolution immunocytochemistry with anti-TRPC1 antibody reveals that these TRPC-encoded SOCs are confined to the PM microdomains adjacent to the underlying 'junctional' ER. Thus, Ca(2+) entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca(2+) stores.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Astrocytes / metabolism*
  • Calcium / metabolism*
  • Calcium Channels / physiology*
  • Cell Membrane / metabolism*
  • Cells, Cultured
  • Cerebral Cortex / metabolism*
  • Endoplasmic Reticulum / metabolism*
  • Fluorescent Dyes
  • Fura-2 / analogs & derivatives
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence / methods*
  • TRPC Cation Channels

Substances

  • Calcium Channels
  • Fluorescent Dyes
  • TRPC Cation Channels
  • transient receptor potential cation channel, subfamily C, member 1
  • Calcium
  • Fura-2